Melanocyte Cell Death Research Articles

Melanocytotoxic chemicals and their toxic mechanisms.

Melanocyte cell death can lead to various melanocyte-related skin diseases including vitiligo and leukoderma. Melanocytotoxic chemicals are one of the most well-known causes of nongenetic melanocyte-related diseases, which induce melanocyte cell death through apoptosis. Various chemicals used in cosmetics, medicine, industry and food additives are known to induce melanocyte cell death, which poses a significant risk to the health of consumers and industrial workers. This review summarizes recently reported melanocytotoxic chemicals and their mechanisms of toxicity in an effort to provide insight into the development of safer chemicals.

Drug delivery of natural products through nano-carriers for effective vitiligo therapy: A compendia review.

Vitiligo is a depigmenting illness that causes white areas on the skin. Vitiligo's pathogenetic genesis is based on the melanocyte's autoimmune destruction, in which oxidative stress causes melanocyte molecular, organelle, and exposure of antigen, as well as melanocyte cell death, and so plays a role in vitiligo progression. Natural compounds have recently shown a wide range of therapeutic bioactivities against a number of skin disorders. The aim of this work is drug delivery of natural products through nano-carriers for effective vitiligo therapy: A compendia review. An online literature analysis was guided for vitiligo therapy, nanotechnology, phytochemical composition, and, types of vitiligo, types of nanomedicine. Appropriate information were taken from different electronic scientific databases such as Web of Science, Science Direct, Elsevier, Google Scholar, Springer, PubMed, and scripts. Nano-carriers-based natural compounds provide a great relationship for the enhancement in the efficacy and safety of pharmacotherapeutic agents for the treatment of vitiligo. In this study focuses on natural compounds' effects and processes on vitiligo models. Although topical therapy plays an important role in vitiligo treatment, its utility and patient compliance are hampered by adverse effects or inadequate efficacy. Novel drug delivery techniques can help improve topical medication delivery by improving epidermal localization, reducing side effects, and increasing effectiveness. This paper covers the significant potential of herbal-derived active compounds as anti-vitiligo drugs, as well as new drug delivery as a viable carrier and future possibilities to investigate.

Plant-Derived Compounds as Promising Therapeutics for Vitiligo.

Vitiligo is the most common depigmenting disorder characterized by white patches in the skin. The pathogenetic origin of vitiligo revolves around autoimmune destruction of melanocytes in which, for instance, oxidative stress is responsible for melanocyte molecular, organelle dysfunction and melanocyte specific antigen exposure as well as melanocyte cell death and thus serves as an important contributor for vitiligo progression. In recent years, natural products have shown a wide range of pharmacological bioactivities against many skin diseases, and this review focuses on the effects and mechanisms of natural compounds against vitiligo models. It is showed that some natural compounds such as flavonoids, phenols, glycosides and coumarins have a protective role in melanocytes and thereby arrest the depigmentation, and, additionally, Nrf2/HO-1, MAPK, JAK/STAT, cAMP/PKA, and Wnt/β-catenin signaling pathways were reported to be implicated in these protective effects. This review discusses the great potential of plant derived natural products as anti-vitiligo agents, as well as the future directions to explore.

Herb Sanqi-Derived Compound K Alleviates Oxidative Stress in Cultured Human Melanocytes and Improves Oxidative-Stress-Related Leukoderma in Guinea Pigs.

Sanqi, a traditional Chinese herb, is widely used for cardiovascular diseases, and its neuroprotective effects against oxidative stress were recently discovered. The purpose of this study was to investigate whether Sanqi-derived compound K (Sanqi-CK), an active metabolite of Sanqi, could protect melanocytes from oxidative stress. Cultured human primary skin epidermal melanocytes (HEMn-MPs) were treated with hydrogen peroxide (H2O2) in the presence or absence of Sanqi-CK. Sanqi-CK exhibited protective effects against H2O2-induced cell death by reducing oxidative stress. In addition, treatment with Sanqi-CK reversed the decreased glutathione reductase activity and decreased ratio of reduced glutathione (GSH)/oxidized glutathione (GSSG) seen in H2O2-treated melanocytes. Furthermore, topical application of Sanqi-CK alleviated leukoderma in guinea pigs, a disorder characterized by melanocyte cell death resulting from rhododendrol-induced oxidative stress. Taken together, these data suggest that Sanqi-CK protects melanocytes against oxidative stress, and its protective effects are associated with modulating the redox balance between GSH and GSSG and activating glutathione reductase. Thus, Sanqi-CK may be a good candidate for preventing melanocyte loss in oxidative-stress-associated pigmentary disorders.

IFN-γ Promotes Apoptosis of Melanocytes in Vitiligo

Vitiligo is an immune-mediated inflammatory condition characterized by selective destruction of epidermal and or follicular melanocytes. It is well established that cell-mediated immunity has been implicated in the pathogenesis of this common skin disorder. Although abnormalities in different subpopulations of T cells may play a role in induction and development of vitiligo, great attention has been paid to CD4+ Th1 cells and cytotoxic CD8+ T lymphocytes (CTLs) (1, 2). The significance of both subgroups in vitiligo development is supported by different observations. For instance, CD8+ T cell-mediated melanocyte destruction has been demonstrated in vitiligo. In addition, an increased accumulation of CD8+ T cells in peripheral blood mononuclear cells (PBMCs) and perilesional skin of patients with progressive vitiligo has been reported (1, 3). On the other hand, elevated expression of Th1 cell specific transcription factor (T-bet) and cytokine (IFNγ) was indicated in PBMCs of patients with vitiligo (2). The exact mechanism by which these cells exert their cytotoxic effects on melanocytes is not well understood. Nonetheless, the results of different laboratory and histological examinations support the hypothesis that apoptosis is an important mechanism for melanocyte disappearance. Apoptosis is a complex process and can be initiated through intrinsic and extrinsic pathways. These pathways are activated by a variety of factors such as cytokines, certain chemicals and other molecular mechanisms. Amongst these factors, cytokines appear to play a pivotal role in promoting apoptotic melanocyte cell death. Cytokines are small secreted proteins that participate in cellular communication and many immunologic functions. Abundant evidence indicated cytokine imbalance (such as IFN-γ, IL-10, IL-17) in the skin and circulation of patients with vitiligo. Therefore, disturbed expression of these molecules may be involved in disease pathogenesis. IFN-γ is a proinflammatory cytokine mainly produced by Th1 lymphocytes, NK cells and CD8+ cytotoxic T cells. This cytokine plays a major role in both innate and adaptive immune response. It can also affect the action of other cytokines in a synergistic or antagonistic manner. The exact function(s) of IFN-γ in the pathology of vitiligo is not known and it may act via different mechanisms. For instance, IFN-γ may participate in the homing of CD8+ T cells to the skin through local induction of chemokines and expression of adhesion molecules on endothelial cells (4). Cytotoxicity via induction of apoptosis has been considered as a major effector mechanism of autoreactive CTLs. Therefore, recruited CD8+ T cells may specifically induce autologous melanocyte apoptosis resulting in disappearance of melanocytes (3). On the other hand, CD4+ Th1 cells (one of the main sources of IFN-γ production) are required for optimal induction of antigen-specific CD8+ T cell effector responses. CD8+ T cells can be considered as a main effector cell of Th1 immunity (5). Therefore, cooperation between CD4+ and CD8+ T cells plays an important role in complete melanocyte clearance. In addition, IFN-γ may influence melanin production. For instance, treatment of melanocytes with IFNγ resulted in significant suppression of melanin production and reduction of mRNA expression levels of tyrosinase and microphthalmia-associated transcription factor (MITF), which regulates differentiation and development of melanocytes and retinal pigment epithelium (6). Moreover, Natarajan et al. indicated a regulatory role of IFN-γ in skin pigmentation. They showed that IFN-γ signaling impedes maturation of the melanosome, which is a major site for synthesis and storage of melanine (7). Altogether, the actual mechanism of melanocytes killing in the skin of patients with vitiligo has not been definitively determined. Current studies indicate a potential role of Th1 and CTL subpopulations in vitiligo pathology as well as a link between IFN-γ and melanocyte abnormalities in this disease. However, further studies are needed to unravel the exact mode of IFN-γ action. A deeper understanding of these mecha-

Abnormal differentiation of dopaminergic neurons in zebrafish trpm7 mutant larvae impairs development of the motor pattern

Transient receptor potential, melastatin-like 7 (Trpm7) is a combined ion channel and kinase implicated in the differentiation or function of many cell types. Early lethality in mice and frogs depleted of the corresponding gene impedes investigation of the functions of this protein particularly during later stages of development. By contrast, zebrafish trpm7 mutant larvae undergo early morphogenesis normally and thus do not have this limitation. The mutant larvae are characterized by multiple defects including melanocyte cell death, transient paralysis, and an ion imbalance that leads to the development of kidney stones. Here we report a requirement for Trpm7 in differentiation or function of dopaminergic neurons in vivo. First, trpm7 mutant larvae are hypomotile and fail to make a dopamine-dependent developmental transition in swim-bout length. Both of these deficits are partially rescued by the application of levodopa or dopamine. Second, histological analysis reveals that in trpm7 mutants a significant fraction of dopaminergic neurons lack expression of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Third, trpm7 mutants are unusually sensitive to the neurotoxin 1-methyl-4-phenylpyridinium, an oxidative stressor, and their motility is partially rescued by application of the iron chelator deferoxamine, an anti-oxidant. Finally, in SH-SY5Y cells, which model aspects of human dopaminergic neurons, forced expression of a channel-dead variant of TRPM7 causes cell death. In summary, a forward genetic screen in zebrafish has revealed that both melanocytes and dopaminergic neurons depend on the ion channel Trpm7. The mechanistic underpinning of this dependence requires further investigation.

Abstract LB-18: BRG1 promotes melanoma survival in response to DNA damage

Abstract The objective of this study is to elucidate the mechanisms by which BRG1 promotes melanoma survival in response to DNA damage. Cutaneous malignant melanoma, is an aggressive malignancy that arises from the transformation of epidermal melanocytes and is notoriously chemoresistant. Exposure to ultraviolet radiation (UVR) from sunlight is the most critical environmental agent involved in melanoma genesis. Melanocytes protect surrounding skin cells from UVR damage and because of this protective role, melanocytes have evolved to be particularly resistant to apoptosis when exposed to UVR. The lineage specific mechanisms that prevent cell death of melanocytes exposed to UVR are thought to contribute to melanoma chemoresistance. Microphthalmia-associated transcription factor (MITF), a master regulator of differentiation in melanocytes, is a lineage addiction oncogene in melanoma. We previously demonstrated that MITF interacts with the SWI/SNF complex to activate MITF target gene expression. SWI/SNF enzymes are multi-subunit ATP dependent chromatin remodeling complexes that regulate gene expression and promote DNA repair. Several of the subunits act as tumor suppressors and are mutated or absent in many forms of cancer. Re-expression of SWI/SNF subunits in cancer cell lines deficient in a particular subunit often results in cell cycle arrest and reversion of the tumorigenic phenotype. However, re-expression of the SWI/SNF subunit, BRG1, in BRG1 deficient melanoma cells increases survival to cisplatin and to UVR exposure. Furthermore, we have found that BRG1 is highly expressed in melanoma tumors and its expression increases during late stages of melanoma, potentially contributing to the chemoresistance typical of advanced melanoma. Our hypothesis is that BRG1promotes lineage specific activation of anti-apoptotic pathways through interactions with MITF. Data indicate that BRG1and MITF expression are closely correlated with expression of the melanoma inhibitor of apoptosis (ML-IAP) gene and that BRG1 promotes dramatic chromatin modifications on the ML-IAP regulatory region, strongly activating expression of this MITF target gene. ML-IAP is a potent inhibitor of apoptosis, which is highly expressed in melanomas and contributes to the survival of melanoma cells exposed to DNA damaging agents. Our current studies are focused on the mechanisms by which BRG1 activates ML-IAP expression and on elucidating the role of ML-IAP in mediating BRG1 induced survival of melanoma cells exposed to UVR. We expect that down-regulation of ML-IAP will significantly compromise BRG1 mediated survival of melanoma cells exposed to UVR. The results from these studies should illuminate a lineage specific mechanism by which BRG1 promotes survival of melanoma cells exposed to a DNA damaging agent and implicate BRG1 as a target for epigenetic based therapeutics to treat this highly resistant malignancy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-18. doi:10.1158/1538-7445.AM2011-LB-18

Potential Redox-Sensitive Akt Activation by Dopamine Activates Bad and Promotes Cell Death in Melanocytes

Dopamine (DA) is a well known oxidative neurotoxin. In addition, Akt has been reported to deliver a survival signal that inhibits apoptosis. However, it has also been reported that chronic Akt activation leads to apoptosis in response to oxidative stress. The objective of the present study was to investigate the possible role of the Akt pathway in vitiligo and its possible relationship with DA-induced cell death using Mel-Ab cells. Cultured Mel-Ab cells were treated with DA with and without N-Acetyl-L-cysteine (NAC), which is known to have antioxidative properties. Cell viability was then assessed by a crystal violet assay and Annexin staining was performed. The changes in the expression of Akt were analyzed by western blot analysis. The cell viability was reduced by approximately 60% in response to treatment with 500 µM DA, and NAC effectively prevented this cytotoxic effect. Likewise, treatment with DA produced numerous Annexin positive cells, while treatment with NAC prevented this apoptotic cell death. Akt was slowly phosphorylated after treatment with DA, while NAC clearly inhibited the DA-induced Akt activation. Western blot analysis also showed that treatment with DA induced the activation of Bad. Finally, LY294002 exerted a protective effect against DA-induced apoptotic cell death. DA may induce redox-sensitive Akt activation and increase the level of Bad, which can promote cell death by heterodimerization with survival proteins. Moreover, NAC effectively protects against DA-induced melanocyte death via inhibition of DA-induced Akt activation.

Tyrosinase-related proteins suppress tyrosinase-mediated cell death of melanocytes and melanoma cells

The synthesis of melanin intermediates through tyrosinase (TYR) involves the production of cytotoxic free radicals. By using recombinant adenoviruses that express TYR, tyrosinase-related protein 1 (TYRP1) or DOPAchrome tautomerase (DCT), we analyzed the biological function of these proteins with regard to melanin production and the growth of melanocytes, fibroblasts, melanoma cells and nonmelanoma cancer cells. High-level expression of TYR produced newly synthesized melanin and induced cell death in all of these cells. However, when TYRP1 or DCT was coexpressed with TYR in melanocytes and melanoma cells, TYR-mediated cell death was clearly decreased. This decrease was not observed in nonmelanocytic cells. Western blot analysis and measurement of enzyme activity revealed that the expression of TYRP1 or DCT had little effect on the amount or activity of cointroduced TYR in either the melanocytic or nonmelanocytic cells. In cells expressing both TYR and TYRP1 or TYR and DCT, the total amount of melanin and/or eumelanin increased substantially more than that in cells expressing TYR alone. On the other hand, the level of pheomelanin was similar in these three cell types. These findings suggest that TYRP1 and DCT play an important role in suppressing TYR-mediated cytotoxicity in melanocytic cells without decreasing TYR expression and/or activity. These biological activities of TYRP1 and DCT may work through the interaction with TYR in melanosomal compartment.

Nitric oxide is toxic to melanocytes in vitro.

Nitric oxide is a diffusible gaseous mediator generated from l-arginine by inducible and constitutive nitric oxide synthases. It has been associated with cytotoxic effects. Inflammatory cells and Langerhans cells can express the inducible form of nitric oxide synthase and produce large quantities of nitric oxide. The proximity of these cells to melanocytes could result in melanocyte cell death. We studied melanocyte susceptibility to nitric oxide using the nitric oxide donor compound sodium nitroprusside and nitric oxide released by the Langerhans like cell-line XS-52 following stimulation with lipopolysaccharide (LPS). Melanocyte lysis, quantified by chromium release in the presence of sodium nitroprusside was both time and concentration dependent. Co-culture of LPS-stimulated XS cells with melanocytes also resulted in melanocyte cell death. No cell death was observed when melanocytes alone were exposed to LPS. Melanocytes were killed even when the co-cultures were performed across Transwells in which there was no direct contact between XS cells and melanocytes. XS-induced melanocyte death was thus dependent on a diffusible factor consistent with nitric oxide. Cell death was markedly decreased in co-cultures performed in the presence of hemoglobin, a nitric oxide quencher. The possible role that nitric oxide may play in disorders associated with loss of pigmentation is discussed.

Tyrp1 and oculocutaneous albinism type 3.

Tyrosinase-related protein 1 (Tyrp1) is a melanocyte-specific gene product involved in eumelanin synthesis. Mutations in the mouse Tyrp1 gene are associated with brown pelage, and in the human TYRP1 gene with oculocutaneous albinism type 3 (OCA3). In the murine system, Tyrp1 expresses significant dihydroxyindole carboxylic acid oxidase (i.e. DHICA oxidase) activity. However, in humans, TYRP1 is enigmatic in that despite extensive efforts focused on the study of its function, its actual role in the human melanocyte is still unclear. There is mounting evidence demonstrating that in addition to its role in eumelanin synthesis, Tyrp1 is involved in maintaining stability of tyrosinase protein and modulating its catalytic activity. Tyrp1 is also involved in maintenance of melanosome ultrastructure and affects melanocyte proliferation and melanocyte cell death. The current review is an attempt to consolidate our understanding of the role of Tyrp1 in the melanocyte.

Up-Regulation and redistribution of Bax in ultraviolet B-irradiated melanocytes.

Ultraviolet B (UVB) radiation may activate or deteriorate cultured human epidermal melanocytes, depending on the doses and culture conditions. It is also reported that cultured human epidermal melanocytes derived from different pigmentary phenotypes showed different responses to UVB radiation. In this study, we examined whether apoptosis of melanocytes can be induced by physiologic doses of UVB irradiation using cultured human epidermal melanocytes derived from oriental males of skin types III and IV. Propidium iodide staining for DNA condensation and flow cytometric analyses demonstrated the apoptotic cell death of melanocytes following UVB irradiation (0-30 mJ/cm2). The levels of p53, Bax, and Bcl-2, determined by immunoblotting, revealed a dose-dependent increase in p53 and Bax, but the level of Bcl-2 remained unchanged. Confocal microscopic examination showed that Bax moved from a diffuse to a punctate distribution after UVB irradiation. However, there were no changes in the pattern of distribution of Bcl-2. These data suggest that the high constitutional level of Bcl-2 may protect melanocytes from UVB-induced injury, and that apoptotic death of melanocytes may be induced by the elevation and redistribution of Bax.

Sulfur Containing Tyrosine Analogs Can Cause Selective Melanocytotoxicity Involving Tyrosinase-Mediated Apoptosis

Sulfur-containing tyrosine analogs such as 4-S-cysteaminylphenol (4-S-CAP) and its N-acetyl derivative, N-acetyl-4-S-CAP, are tyrosinase substrates and can cause selective cytotoxicity or cell death of melanocytes and melanoma cells. It is not clear, however, if the cytotoxicity derives from a cytostatic or cytocidal effect. The latter can also be either apoptotic or necrotic. This paper summarizes our attempt to clarify the nature of melanocytotoxicity and cell death by using a new derivative of 4-S-CAP, N-propionyl-4-S-CAP (NPr-CAP). The i.p. administration of NPr-CAP caused marked depigmentation of black hair follicles in C57 mice. At 12 h postadministration of NPr-CAP, follicular melanocytes showed histochemical and morphologic features indicative of apoptosis by TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining and electron microscopy. The agarose gel electrophoresis of DNA from drug-treated melan a2 cells, an immortal melanocyte line of C57 black mice, showed the nucleosomal DNA ladder pattern. NPr-CAP caused irreversible cytotoxicity in melan a2 and the effect was inhibited by a tyrosinase inhibitor, phenylthiocarbamide. The tyrosinase-mediated cytotoxicity of NPr-CAP was further confirmed by the decreased viability of COS 7 monkey-kidney cells, which expressed a level of high tyrosinase activity through transfection of human tyrosinase cDNA. NPr-CAP, however, also transiently inhibited the proliferation of melan c cells, a control tyrosinase-negative albino melanocyte line, and vector-transfected COS 7 cells. Thus, the major process of NPr-CAP-mediated melanocytotoxicity involves cytocidal apoptosis associated with active tyrosinase. In addition, there is transient, nontyrosinase-mediated cytostatic cytotoxicity.

Biological role of tyrosinase related protein and its biosynthesis and transport from TGN to stage I melanosome, late endosome, through gene transfection study.

Tyrosinase-related protein (TRP)-1 is one of the most abundant melanosomal glycoproteins involved in melanogenesis. This report summarizes our recent research efforts related to the biological role and biosynthesis of TRP-1 and its transport from TGN (trans-Golgi network) to the stage I melanosome. Our UV irradiation and tyrosinase and TRP-1 cDNA co-transfection studies indicated that human TRP-1 is involved in not only melanogenesis but also prevention of melanocyte death, which may occur during biosynthesis of melanin pigment in the presence of tyrosinase. Furthermore, a coordinated gene interaction was indicated between tyrosinase and TRP-1, resulting in upregulation of mRNA and protein expression of LAMP (lysosome-associated membrane protein)-1 that would directly prevent the tyrosinase-mediated programmed cell death of melanocytes. Similar to tyrosinase, however, TRP-1 appears to require a molecular chaperone, calnexin, which we have cloned recently. Our cDNA transfection study of tyrosinase with calnexin showed clearly the necessity of calnexin in order to have efficient, functional activity of melanosomal glycoprotein, especially tyrosinase. Once glycosylation is completed, TRP-1 will be transported from TGN to the stage I melanosome. At this stage, TRP-1 will have its own target signal, in particular, tyrosine-rich leucine residues in cytoplasmic tail. Our TRP-1 cDNA transfection and immunoelectron microscopy study shows that TRP-1 will be transported through small vesicles, probably non-clathrin-coated type, to large vacuoles, identical to the MPR (mannose-6-phosphate receptor)-positive, late endosomes. In this transport process a low molecular weight G-protein, rab-7, was isolated from the purified melanosomal protein on 2D-PAGE and identified by subsequent sequencing and PCR amplification. Confocal microscopy with double immunostaining and immunoelectron microscopy confirmed the co-localization of rab-7 and TRP-1 in the melanosomes with early stages of maturation (I-HI). Furthermore, this process will also be regulated by phosphatidylinositol 3-kinase (PI-3 kinase).

Premature Avian Melanocyte Death Due to Low Antioxidant Levels of Protection: Fowl Model for Vitiligo

Feather melanocytes in the Barred Plymouth Rock (BPR) and White Leghorn (WL) chickens die prematurely in vivo when compared to the wild type Jungle Fowl (JF) chicken. Since these mutant melanocytes live in vitro, an environmental factor in the feather must precipitate their death. Results show that the addition of selected antioxidants, glutathione (GSH) and superoxide dismutase (SOD), can rescue these mutant melanocytes in vitro that have been placed under stress conditions that cause their premature cell death. Measurements of in vivo levels of GSH, catalase, and SOD show no significant difference in catalase activity between the JF, BPR, and WL feathers but do show a significant reduction in GSH activity in both the BPR and WL feathers to approximately 66% of the GSH concentration found in JF feathers. SOD activity in the BPR tissue is reduced significantly to approximately 50% of the JF activity and the WL SOD activity is reduced significantly to approximately 50% of the BPR SOD activity. Preliminary results of measurements of glutathione peroxidase activity indicate there is no difference in the levels of this enzyme in JF, BPR and WL feathers. A working hypothesis, based on current results, is proposed for premature cell death in BPR and WL feather melanocytes. The BPR melanocytes are genetically sensitive due to a defect in their SOD and GSH levels caused by the barring gene (B) and their death, due to reactive species of oxygen radicals, is precipitated in the poorly vascularized feather by the accumulation of oxygen radicals due to the low turnover of tissue fluids. The WL chicken carries the dominant white gene (I) in addition to the B gene. This gene directs the further reduction of the level of SOD and, when combined with the cell death mechanism already present in the BPR chicken, causes the WL feather melanocytes to die much earlier than the BPR feather melanocytes which in turn die much earlier than the wild type JF melanocytes. This same mechanistic hypothesis could apply as a cause of premature melanocyte cell death in human vitiligo wherein the vitiliginous melanocytes may have a genetic defect in their oxygen radical protection system.